Oxalic acid, a compound that is toxic to almost all organisms (Hodgkinson, 1977), plays several important roles in fungal growth and metabolism (Dutton et al., 1996), and in biological mechanisms underlying fungal pathogenesis. For example, Aspergillus niger, which can colonize lung tissue in immunocompromised individuals, excretes enough oxalate to form crystalline calcium salts as part of necrotizing otomycosis (Landry et al., 1993) and, in certain cases, can give rise to fatal pulmonary oxalosis (Kimmerling et al., 1992; Metzger et al., 1984). A number of enzymes have evolved in plants (oxalate oxidase) (Kotsira et al., 1997), fungi (oxalate decarboxylase) (Lillehoj et al., 1965) and bacteria (oxalyl-CoA decarboxylase) (Quayle, 1963) to remove oxalate from the environment. Oxalate decarboxylase (OxDC) catalyzes a remarkable transformation in which the C—C bond in oxalate is cleaved to give carbon dioxide and formate: 
The enzyme is presumably important in fungal metabolism as a protective agent against internalization of neutral oxalate formed in the environment as the soil pH drops due to wood degradation or secretion of oxalic acid. Oxalate decarboxylase was first isolated from basidiomycete fungi (Shimazono, 1955), and has subsequently been identified in several species of filamentous fungi, including Myrothecium verrucaria (Lillehoj et al., 1965), certain strains of Aspergillus niger (Emiliani et al., 1964) and Flammulina velutipes (Mehta et al., 1991), and the common button mushroom Agaricus bisporus (Kathiara et al., 2000). OxDC expression can also be induced in the white-rot fungus Coriolus versicolor (Shimazono et al., 1957), and very recent work has also shown that OxDC is present in Bacillus subtilis (Tanner et al., 2000), although this appears to be the only bacterium in which the presence of this enzyme has been unambiguously demonstrated. While it has been demonstrated that the bacterial OxDC is manganese-dependent (Tanner et al., 2001), the detailed catalytic mechanism by which oxalate is converted to formate and carbon dioxide has not yet been elucidated.
Early experiments employing the Aspergillus niger OxDC showed that (i) enzymatic CO2 evolution requires oxalate to the exclusion of other biologically relevant carboxylic acids, (ii) oxygen is required for catalytic turnover, although high oxygen tensions inhibit the enzyme (Emiliani et al., 1968), and (iii) a sub-stoichiometric quantity of oxygen is converted to hydrogen peroxide during the reaction. Weak reductants such as phenylenediamines and diphenols activate the enzyme, whereas treatment with strong reductants such as dithionite and hydroxylamine eliminate OxDC activity. No evidence was found for the presence of exogenous cofactors in the native Aspergillus niger OxDC, and the enzyme was reported not to contain iron and copper ions as purified. A general, oxygen-dependent, mechanism involving the formation of free radical species was proposed to account for these experimental observations (Emiliani et al., 1968). In light of the demonstrated dependence of OxDC activity on dioxygen (Tanner et al., 2001), a hypothetical catalytic mechanism is currently favored in which bound manganese undergoes an oxidation to give a species capable of abstracting an electron directly from oxalate to give the radical anion 1 (Scheme 1A). It is also likely that oxalate binding to manganese precedes that of dioxygen. C—C bond cleavage, which might be expected to be a fast chemical step, then yields 2 and concomitant proton and electron transfer (Su et al., 1998) to give formate regenerates the oxidized metal species. No evidence for the involvement of a redox-active co-factor (Halcrow, 2001) is provided by the recent crystal structure of the bacterial OxDC (Anand et al., 2002). The structures of the fungal and bacterial oxalate decarboxylases are likely to be very similar on the basis of sequence identity and the likely evolutionary relationship between the two enzymes (Dunwell et al., 2000). The observed correlation between H2O2 formation and pO2 in the OxDC-catalyzed reaction (Emiliani et al., 1968) is consistent with such a mechanism if oxidation of the formyl radical anion 2 takes place to generate CO2, peroxide anion and an inactive form of OXDC (Scheme 1B). 
Although it has been speculated that Mn(III) and Mn(IV) are the redox active forms of the metal during catalysis (Anand et al., 2002), there is no published evidence to support such a claim. Equally, the intermediacy of a protein-based radical cannot be ruled out on the basis of current biochemical and structural information on Bacillus subtilis OxDC. This proposal has the merit of rationalizing the observed correlation between the amounts of hydrogen peroxide formed under the assay conditions and the partial pressure of oxygen. Chemical precedent for a mechanism involving radical-dependent decarboxylation of oxalate has been obtained in model chemical studies (Drummond et al., 1953; Halliwell, 1972), including direct electron-nuclear double resonance (ENDOR) observation of formate radical produced by irradiation of oxalate crystals (Edlund et al., 1973). Additional support is provided by the Kolbe reaction in which one-electron electrochemical oxidation of carboxylic acids results in production of CO2 and daughter radicals (Bard et al., 1978). Nevertheless, a radical-based mechanism for OxDC-catalyzed oxalate degradation would gain considerable credence upon observation of paramagnetic species formed on incubation of the enzyme with substrate.
Kidney-urinary tract stone disease (urolithiasis) is a major health problem throughout the world. Most of the stones associated with urolithiasis are composed of calcium oxalate alone or calcium oxalate plus calcium phosphate. Other disease states have also been associated with excess oxalate. These include, vulvodynia, oxalosis associated with end-stage renal disease, cardiac conductance disorders, Crohn's disease, and other enteric disease states.
Oxalic acid (and/or its salt-oxalate) is found in a wide diversity of foods, and is therefore, a component of many constituents in human and animal diets. Increased oxalate absorption may occur after foods containing elevated amounts of oxalic acid are eaten. Foods such as spinach and rhubarb are well known to contain high amounts of oxalate, but a multitude of other foods and beverages also contain oxalate. Because oxalate is found in such a wide variety of foods, diets that are low in oxalate and which are also palatable are hard to formulate. In addition, compliance with a low oxalate diet is often problematic.
Normal tissue enzymes also produce endogenous oxalate metabolically. Oxalate (dietary oxalate that is absorbed as well as oxalate that is produced metabolically) is not further metabolized by tissue enzymes and must therefore be excreted. This excretion occurs mainly via the kidneys. The concentration of oxalate in kidney fluids is critical, with increased oxalate concentrations causing increased risk for the formation of calcium oxalate crystals and thus the subsequent formation of kidney stones.
The risk for formation of kidney stones revolves around a number of factors that are not yet completely understood. Kidney-urinary tract stone disease occurs in as much as 12% of the population in Western countries and about 70% of these stones are composed of calcium oxalate or of calcium oxalate plus calcium phosphate. Some individuals (e.g., patients with intestinal disease such as Crohn's disease, inflammatory bowel disease, or steatorrhea and also patients that have undergone jejunoileal bypass surgery) absorb more of the oxalate in their diets than do others. For these individuals, the incidence of oxalate urolithiasis increases markedly. The increased disease incidence is due to increased levels of oxalate in kidneys and urine, and this, the most common hyperoxaluric syndrome in man, is known as enteric hyperoxaluria. Oxalate is also a problem in patients with end-stage renal disease and there is recent evidence (Solomons et al, 1991) that elevated urinary oxalate is also involved in vulvar vestibulitis (vulvodynia).
Bacteria that degrade oxalate have been isolated from human feces (Allison et al., 1986). These bacteria were found to be similar to oxalate-degrading bacteria that had been isolated from the intestinal contents of a number of species of animals (Dawson et al., 1980; Allison et al., 1981; Daniel et al., 1987). These bacteria are different from any previously described organism and have been given both a new species and a new genus name (Allison et al., 1985).
Not all humans carry populations of O. formigenes in their intestinal tracts (Allison et al., 1995; Doane et al, 1989). There are low concentrations or a complete lack of oxalate degrading bacteria in the fecal samples of persons who have had jejunoileal bypass surgery (Allison et al., 1986). Also, certain humans and animals may maintain colonies of O. formigenes but nevertheless have excess levels of oxalate for reasons that are not clearly understood.
U.S. Pat. No. 6,355,242 and published international patent application WO 98/52586 disclose delivery of bacteria and/or oxalate-degrading enzymes to intestinal tracts of persons or animals, thereby reducing oxalate in the intestinal tract of those persons or animals who are at risk for oxalate related disease.
OxDC of Aspergillus niger, which converts oxalate directly to formate and carbon dioxide without the need for exogenous co-factors, can provide a therapeutic approach at a significant reduction in cost. A second benefit of using Aspergillus niger OxDC is that the enzyme has a pH-optimum of 4.2, making it useful for oxalate degradation in the upper intestine. Since Aspergillus niger is also used in the production of citrate, which is then added to food products and dietary supplements, it is likely that no significant side effects will be observed when this form of OxDC is administered in the human gastrointestinal tract.